Table 1. Primers, annealing temperatures, and product sizes for PCR amplification.

Transcription

1 Table 1. Primers, annealing temperatures, and product sizes for PCR amplification. Gene Direction Primer sequence (5 3 ) Annealing Temperature Size (bp) BRCA1 Forward TTGCGGGAGGAAAATGGGTAGTTA 50 o C 292 Reverse TGTGCCAAGGGTGAATGATGAAAG REF1/APE1 Forward ACAAAGAGGCAGCAGGAGAG 53 o C 532 Reverse GAGGTCTCCACACAGCACAA NTH1 Forward CAGGTGCTGCTGTCACTGAT 53 o C 168 Reverse CACCTTGCTCCTCCAGAAAC OGG1 Forward GCAGCAGCTACGAGAGTCCT 55 o C 227 Reverse TTCCCAGTTCCTTGTTGGTC XRCC1 Forward GATTCTGGGGACACAGAGGA 52 o C 216 Reverse AGGGAACTCCCCGTAAAGAA Ligase III Forward TTCCCCAACTCAAGGAACTG 50 o C 245 Reverse CTTCATAGCGGAAGGCTTTG Catalase Forward GCCTGGGACCCAATTATCTT 51 o C 203 Reverse GAATCTCCGCACTTCTCCAG SOD1 Forward AGGGCATCATCAATTTCGAG 50 o C 465 Reverse ACATTGCCCAAGTCTCCAAC SOD2 Forward CTGGACAAACCTCAGCCCTA 52 o C 199 Reverse CTGATTTGGACAAGCAGCAA β-actin Forward AAATCTGGCACCACACCTTC 49 o C 432 Reverse CCATCTCTTGCTCGAAGTCC

2 Table 2. Sequences of oligonucleotides for cleavage and ligase assays. Assay Oligo Type Sequences of the oligonucleotides (5 3 ) Product size (bp) 8-OxoG Wild-type 5 FITC-ATACGCATATACCGCT (G) TCGGCCGATCTCCGAT Modified 5 FITC-ATACGCATATACCGCT (8-OxoG) TCGGCCGATCTCCGAT 17 Complementary 5 ATCGGAGATCGGCCGA (C) GGCGGTATATGCGTAT Thymine Wild-type 5 FITC-GAACGACAGA (T) GACACGACAGACAAGCA Glycol Modified 5 FITC-GAACGACAGA (Thymine Glycol) GACACGACAGACAAGCA 11 Complementary 5 TGCTTGTCTGTCGTGTC (A) TCTGTCGTTC REF1/APE1 Wild-type 5 FITC-ATATACCGCGG (C) CGGCCGATCAAGCTTATT Modified 5 FITC-ATATACCGCGG (AP) CGGCCGATCAAGCTTATT 12 Complementary 5 AATAAGCTTGATCGGCCG (G) CCGCGGTATAT Ligase III LigIII-1 GCATATGGAAATCGTCAGTTAGGAGCACTC LigIII-2 [γ- 32 P]-5'TAGCCTGAAGTGCATGCCTAGCACGTTCTACCCAGTTCCG 40 Complementary 5 CGGAACTGGGTAGAACGTGCTAGGCATGCACTTCAGGCTAGAGTG CTCCTAACTGACGATTTCCATATGC

3 LEGENDS FOR SUPPLEMENTAL FIGURES: Fig. S1. BRCA1 regulates DNA cleavage at a thymine glycol (TG) site in T47D cells. A-C. Proliferating cells were transiently transfected overnight with vehicle, wild-type BRCA1 (wtbrca1), or empty pcdna3 vector, washed, and allowed to recover for several hours prior to assay. Nuclear extracts were prepared; and 30 μg aliquots were tested for their ability to incise duplex oligonucleotides containing a TG lesion, using the corresponding wild-type duplex oligonucleotide as a control. Panel A shows a representative DNA gel; while panel B shows the quantitative extents of cleavage, expressed as means ± SEMs of three independent experiments. The protein levels of BRCA1, NTH1, and α-actin for the different transfection conditions are shown in panel C. D-F. T47D cells were treated with BRCA1- sirna (100 nm), control (CON)-siRNA, or vehicle only and assayed for incising activity at a TG site as described above. Panel D shows a representative DNA gel; while panel E shows the quantitative extent of cleavage. The BRCA1, NTH1, and actin protein levels for the different treatment conditions are shown in panel F. In B and E, an asterisk represents a statistically significant difference (P < 0.05, two-tailed t-test). Fig. S2. BRCA1 regulates DNA cleavage at an 8-oxoguanine (8-OxoG) site in T47D cells. A-C. Proliferating T47D cells were transiently transfected overnight with vehicle only, wtbrca1, or empty pcdna3 vector, washed, and allowed to recover for several hours prior to assay. Nuclear extracts were prepared; and 100 μg aliquots were tested for their ability to incise duplex oligonucleotides containing an 8-OxoG lesion, using the corresponding wild-type duplex oligo as a negative control. Panel A shows a representative DNA gel; while panel B shows the quantitative extents of cleavage, expressed as means ± SEMs of three independent experiments. Western blots showing the protein levels of BRCA1, OGG1, and α-actin (control for loading and transfer) for the different transfection conditions are provided in panel C. D-F. T47D cells were treated with BRCA1-siRNA (100 nm), control (CON)-siRNA (100 nm), or vehicle only for 72 hr, after which nuclear extracts were prepared and assayed for incising activity as above. Panel D shows a representative DNA gel; while panel E shows the quantified extent of cleavage, expressed as means ± SEMs of three independent experiments. The protein levels of BRCA1, OGG1, and α-actin for the different treatment conditions are shown in panel F. *P < 0.05 (two-tailed t-test). Fig. S3. A point mutant BRCA1 protein does not stimulate OGG1 incising activity or expression. A- C. Proliferating T47D cells were transiently transfected overnight with vehicle only, empty pcdna3 vector, or BRCA1 mutant T300G ( 61 Cys Glyc); washed; and allowed to recover for several hours prior to assay. Nuclear extracts were prepared; and 100 μg aliquots were tested for their ability to incise duplex oligonucleotides containing an 8-OxoG lesion, using the corresponding wild-type duplex oligo as a negative control. Panel A shows a representative DNA gel; while panel B shows the quantitative extents of cleavage, expressed as means ± SEMs of three independent experiments. Western blots showing the protein levels of BRCA1, OGG1, and α-actin (loading control) for the different transfection conditions are provided in panel C. Fig. S4. BRCA1 regulates DNA cleavage at an apyrimidinic (AP) site in T47D cells. A-C. T47D cells were transfected with vehicle, wtbrca1, or empty pcdna3 vector; and aliquots of nuclear extracts (50 μg) aliquots were assayed for incising activity around an AP site in a duplex oligonucleotide. Panel A shows a representative assay; while panel B shows the quantification of cleavage (based on three independent experiments) and panel C shows the BRCA1, REF1/ APE1, and α-actin protein levels for the different transfection conditions. D-F. T47D cells were treated with BRCA1-siRNA (100 nm), CONsiRNA, or vehicle and assayed for incising activity as above. Panel D shows a representative DNA gel; while panel E shows the quantitative extent of cleavage. The BRCA1, REF1/APE1, and actin protein levels for the different treatment conditions are shown in F. *P < 0.05 (two-tailed t-test).

4 Fig. S5. Effects of BRCA1 over/under-expression on glycosylase protein levels in MCF-7 cells. This figure shows the effects of BRCA1 over-expression (A, C, E) or under-expression (B, D, F) on the protein levels of NTH1 (A, B), OGG1 (C, D), and REF1/APE1 (E, F) corresponding to the experiments shown in Figs Values are means ± SEMs of at least three independent experiments plotted as the fold change of the protein/actin relative to that for vehicle-treated cells. *P < 0.005, two-tailed t-tests. Fig. S6. Effect of BRCA1 on DNA ligase III activity. MCF-7 or T47D cells were transfected with wtbrca1 (vs pcdna3 vector or vehicle only) or they were treated with BRCA1-siRNA (vs controlsirna or vehicle only), as described above. The nuclei were then isolated and assayed for DNA ligation activity, as described in the Methods section. Panels A-D show representative DNA gels; while panels E- H show the quantified results based on a minimum of three independent experiments per cell line per treatment condition. This assay measures the ability of the nuclear extracts to ligate a labeled 40-mer to an unlabeled 30-mer, to form a labeled 70-mer oligonucleotide. Note: The two left lanes in panel A show the positions of the [γ- 32 P]-labeled 70-mer and [γ- 32 P]-labeled 40-mer oligonucleotides. The left most lanes in panels B, C, and D represent positive control ligation reactions carried out using T4 DNA ligase rather than nuclear extracts. Fig. S7. BRCA1 regulates expression of BER enzymes in T47D cells. A,B. Proliferating T47D cells were transfected overnight with wtbrca1, empty pcdna3 vector, or vehicle only; washed; allowed to recover from the transfection in fresh culture medium; and harvested for semi-quantitative RT-PCR assays, as described in the Methods section. Representative gels showing the amplified bands corresponding to BRCA1, OGG1, NTH1, REF1/APE1, XRCC1, and β-actin are provided in panel A. The PCR bands were quantified by densitometry; and the mrna levels were normalized to β-actin and expressed relative to that of the vehicle control in panel B. The fold-change values are means ± SEMs of at least three independent experiments. *P < 0.05 relative to vehicle control (two-tailed t-test). C,D. T47D cells transfected with expression vectors (C) or treated with sirnas (D) as before were harvested and Western blotted to detect BRCA1, XRCC1, ligase III, or actin (loading control). Fig. S8. Unlike OGG1, BRCA1 does not directly stimulate incision at an 8-OxoG site. A,B. T47D cells were transfected with wtbrca1 or empty pcdna3 vector and either assayed for OGG1 incising activity (A) or Western blotted as described in Fig. S2 legend. C,D. OGG1 incising assays were performed as above, except that the indicated quantity of purified BRCA1 protein (C) or purified OGG1 protein (D), respectively obtained from commercial sources was added to the reaction mixture. Fig. S9. Role of BER enzymes in BRCA1 protection of T47D cells against oxidative stress due to H 2 O 2. A. Proliferating cells in six-well dishes were transfected overnight with the indicated expression vectors (using empty pcdna3 vector to keep the total transfected DNA content constant); washed; post incubated in fresh medium for 24 hr to allow gene expression; harvested; and inoculated into 96-well plates in fresh medium. The cells were allowed to attach and recover for another 24 hr; exposed to the indicated concentration of H 2 O 2 for 24 hr; and tested for cell viability by MTT assays. Values plotted are means ± SEMs of three independent experiments, with each experiment using 8 replicate wells per assay condition. B. Cells in 6-well dishes were treated with BRCA1-siRNA (100 nm), CON-siRNA (100 nm), or vehicle only for 72 hr and then inoculated into 96-well plates for H 2 O 2 survival assays as described above. For combination treatments [BRCA1-siRNA+wtREF1], after incubation of the cells with sirna (100 nm) for 24 hr, they were transfected with wtref1 overnight and allowed to recover for another 24 hr. The cells were then harvested; inoculated into 96-well dishes; and assayed for sensitivity to H 2 O 2 as above. In the left panels, significant increases in survival due to wtbrca1 (relative to pcdna3 vector) or reductions in survival due to BRCA1-siRNA (relative to CON-siRNA) are indicated by an asterisk (*) (P < 0.05). In the right panels, the asterisks represent significant comparisons of (wtbrca1 +DN-REF1) vs DN-REF alone or (BRCA1-siRNA+wtREF1) vs BRCA1-siRNA alone. C-E. Treatments with sirna

5 alone or combinations of sirna plus wtbrca1 vector and subsequent assays of cellular sensitivity to H 2 O 2 were carried out as above. The effects of sirnas for OGG1 (C), NTH1 (D), and REF1 (E) are shown. Cell viability values are means ± SEMs of three independent experiments and are normalized to the control (vehicle only) treatment. Significant reductions in survival due to OGG1-siRNA, NTH1- sirna, or REF1-siRNA (left panels) [relative to control (CON)-siRNA] are indicated by an asterisk (*) (P < 0.05). In right panels, increases in survival due to a combination of BER enzyme-sirna plus wtbrca1 (relative to BER enzyme-sirna alone) are shown by anasterisk (P < 0.05). Fig. S10. Expression of wtref1 and DN-REF1 and efficacy of BER enzyme knockdowns. T47D cells were transfected with wtref1 (A) or DN-REF (B) or treated with REF1-siRNA (C), OGG1-siRNA (D), or NTH1-siRNA (E), as described above. The cells were then harvested for Western blotting to detect the indicated BER enzyme or actin. Fig. S11. Knockdown of NF-YA does not block BRCA1 stimulation of BER enzyme expression. T47D cells were treated with control (CON)-siRNA or NF-YA-siRNA (100 nm x 48 hr) and then transfected overnight with either a wild-type (wt) BRCA1 expression vector (+) or empty pcdna3 vector (-). The cells were post-incubated for 24 hr to allow gene expression and then subjected to Western blotting to detect the indicated proteins. α-actin was used as a control for loading and transfer. Panel A shows a typical Western blot. Panel B shows quantification of these experiments by densitometry. Values plotted are means ± SEMs of the fold-change (ratio of protein/actin normalized to CON-siRNA and empty vector transfection value) based on three independent experiments. Fig. S12. BRCA1 regulates activity of a short (0.4 kb) NTH1 promoter. A. T47D cells were transfected overnight with the indicated NTH1 luciferase (NTH1-Luc) reporter (containing 0.4 kb or 1.2 kb of NTH1 promoter sequence upstream of the translation start site) or the empty pgl3-luc reporter plasmid; post-incubated for 24 hr to allow gene expression; and harvested for luciferase assays. B. T47D cells were co-transfected overnight with wtbrca1 (or empty pcdna3 vector) and with an NTH1-Luc reporter containing 0.4 kb of NTH1 promoter sequence or the empty pgl3-luc reporter plasmid. The cells were then post-incubated for 24 hr to allow gene expression and harvested for luciferase assays. Luciferase values are expressed as fold-change relative to control conditions (=1.0) (pcdna3+nth1- Luc). C. T47D cells were pre-treated with CON-siRNA or BRCA1-siRNA (100 nm x 48 hr); transfected with NTH1-Luc (0.4 kb) or pgl3-luc; and assayed as above. Luciferase values were expressed as foldchange relative to control conditions (=1.0) (CON-siRNA+NTH1-Luc). D. Cells were pre-treated with CON-siRNA or OCT1-siRNA; co-transfected overnight with wtbrca1 (+) or empty vector (-) plus NTH1-Luc (0.4 kb) reporter; post-incubated for 24 hr to allow gene expression; and harvested for luciferase assays. Luciferase values are expressed as fold-change relative to control conditions (=1.0) (CON-siRNA+NTH1-Luc). Luciferase values are means ± SEMs of three independent experiments. P values were calculated using two-tailed t-tests. Fig. S13. BRCA1 over-expression does not stimulate the short NTH1 promoter activity in Oct1 null (Oct1 -/- ) mouse embryo fibroblasts (MEFs). A,B. Wild-type (Oct1 +/+ ) (A) or Oct1 -/- (B) MEFs were cotransfected with wtbrca1 (vs pcdna3 vector) and the NTH1-Luc (0.4 kb) or empty pgl3-luc reporter overnight; post-incubated for 24 hr to allow gene expression; and assayed for luciferase activity. Luciferase values are expressed as fold-change relative to control (=1.0) (pcdna3+nth1-luc). Panel C compares the activities of the NTH1-Luc (0.4 kb) reporter in Oct1 +/+ vs Oct1 -/- MEFs. All values are means ± SEMs of three independent experiments. P values were calculated using two-tailed t-tests. Fig. S14. Oxidative stress due to H 2 O 2 stimulates BRCA1 and BER enzyme expression. A. Subconfluent proliferating T47D cells were treated with H 2 O 2 (250 nm) for 0. 2, or 24 hr and then harvested for Western blotting to detect BRCA1, NTH1, OGG1, REF1/APE1, and actin (loading control).

6 B. T47D cells were treated with H 2 O 2 (250 nm) for 24 hr and then harvested for NTH1 incising activity assays as described in Fig. S1 legend.

7 Fig. S1. A B C D E F

8 Fig. S2. A B C D E F

9 Fig. S3. A B C

10 Fig. S4. A B C D E F

11 Fig. S5. A B C D E F

12 Fig. S6. A B C D E MCF-7 F G H T47D MCF-7 T47D

13 Fig. S7. A B C D

14 Fig. S8. A B C D

15 A B Fig. S9. Saha et al., 2010 C D E

16 Fig. S10. A B C D E

17 Fig. S11. A B

18 Fig. S12. A B P < 0.05 C P < 0.02 D P < 0.05

19 Fig. S13. A B C P < Fold Change Fold Change

20 Fig. S14. A B